As shown in Figure three, A and B, IM 9 cell lines, every single expressing unique shRNAs targeting JAK3 and TYK2, were tested for expression of JAK3 and TYK2 and their capacity to activate NKL and NK 92 cells. Three of 4 JAK3 shRNAs and two of four TYK2 shRNAs success completely lowered expression of the target protein, but none of these shRNAs induced enhanced secretion of IFN from either NKL or NK 92 effector cells. These benefits confirmed that distinct down regulation of JAK1 and JAK2 but not JAK3 or TYK2 could modu late tumor cell susceptibility to NK cell activity. To examine the specificity of JAK1 inhibition on susceptibil ity to NK cell activity, we undertook additional characterization of IM 9 target cells expressing each from the three JAK1 shRNA vectors. As shown in Figure 4A, JAK1 protein expression was reduced in IM 9 cells expressing Jak1 1 and Jak1 3 shRNAs.
These effects were spe cific for JAK1, and expression of JAK2, JAK3, and TYK2 was not decreased. Similarly, quantitative RT PCR demonstrated lowered levels of JAK1 mRNA in these cell lines. As shown in Figure 4C, reduced expression of JAK1 resulted in drastically larger levels of IFN secretion by NKL and NK 92 effector cells. Intracellular staining confirmed selelck kinase inhibitor that IFN was derived from NK effector cells. In traditional cytotoxicity assays, IM 9 cells with reduced expression of JAK1 were a lot more sus ceptible to lysis by both NKL and NK 92 effector cells when com pared with IM 9 cells infected having a manage shRNA. No difference in cytotoxicity was noted in IM 9 cells expressing shRNA Jak1 two that had not impacted JAK1 pro tein expression.
Enhanced killing of JAK1 additional hints knockout IM 9 cells by NK cells was also confirmed making use of an Annexin V assay we developed to quantify the induction of apoptosis in target cells incubated with NK effector cells. Within this assay, effector cells were incubated with target cells at a 1:1 effector/target ratio to get a 12 hour period. As shown in Fig ure 4E, IM 9 cells lacking expression of JAK1 underwent significantly far more apoptosis than IM 9 cells infected with a control hairpin or having a JAK1 shRNA that will not lessen JAK1 expression. Increased apoptosis was observed when IM 9 cells were incubated with either NKL or NK 92 effector cells, but the level of spontaneous apoptosis for IM 9 cells expressing each of your JAK1 shRNAs was generally significantly less than 7% if no effector cells had been present. Results of equivalent experiments carried out with 3 shRNAs specif ic for JAK2 are summarized in Figure 5.
Western blot analysis and quantitative RT PCR confirmed that IM 9 cells expressing Jak2 three and Jak2 4 expressed lower levels of JAK2, and expression of those shRNAs did not affect expression of the other members in the JAK family.